Creatinine iminohydrolase, sometimes referred to as creatinine desimidase, is an enzyme which specifically catalyzes the hydrolysis of creatinine to ammonia. The aerobic soil microorganism ATCC #31546 represents a new microbial source of creatinine iminohydrolase. Creatinine iminohydrolase from this source is particularly useful because it is substantially free from urease activity, urease being a highly undesirable interferent for creatinine assays conducted on aqueous liquids containing both creatinine and urea. Accordingly, urease-free creatinine iminohydrolase together with an ammonia detector can provide a quantitative assay composition for the determination of creatinine contained in aqueous liquids, e.g., serum. The urease-free creatinine iminohydrolase from the aerobic soil microorganism ATCC #31546 is described in greater detail in Goodhue, Esders, and Masurekar copending U.S. patent application Ser. No. 91,218 filed concurrently herewith and entitled "Creatinine Iminohydrolase Free From Urease Activity".
The above-referenced urease-free creatinine iminohydrolase is produced intracellularly by the microorganism and must be extracted therefrom following growth of the microbial cells. A useful fermentation process for growing microbial cells of ATCC #31546 containing substantial quantities of the desired urease-free creatinine iminohydrolase enzyme is described in Masurekar, copending U.S. patent application Ser. No. 91,216 filed concurrently herewith and entitled "Process and Nutrient Medium For Growing Microorganisms".
Recovery of the intracellularly produced creatinine iminohydrolase enzyme referenced hereinabove is carried out by known techniques wherein the cells are disrupted to release the enzyme, followed, if desired, by partial purification of the enzyme. Well-known cell disruption techniques include sonication, grinding, and the like. Partial purification is typically performed by separating the desired enzyme from the microbial cell debris such as by centrifugation, filtration, or the like followed by organic solvent fractional precipitation to separate the desired enzyme from other microbial cell protein.
The aforementioned disruption and partial purification procedure typically employs a sequence of individual process steps including cell disruption and separation of the desired intracellular enzyme from microbial cell debris, followed by organic solvent fractional precipitation. For example, the aforementioned sequence of individual process steps is disclosed in U.S. Pat. Nos. 4,087,329 and 4,134,793 which describe the recovery of creatinine desimidase (another name for creatinine iminohydrolase) from various aerobic microorganisms. This sequential procedure, however, extracts only a portion of the enzyme from the microbial cells. In addition, the individual process step of separating the desired enzyme from microbial cell debris requires extensive centrifugation or filtration equipment and time when carried out on a large-scale production basis.
Moreover, creatinine iminohydrolase produced intracellularly by an aerobic soil microorganism, when recovered from the microorganism by the aforementioned sequential procedure, exhibits a problem of enzyme instability. That is, the enzyme, during recovery from the microbial cells, often exhibits a marked decrease in activity. This same enzyme instability problem may also be exhibited by other intracellular enzymes.
Accordingly, a more efficient process for recovering enzymes produced intracellularly by aerobic microorganisms would clearly be a useful contribution to the art. Such a process would be particularly desirable for use with intracellular enzymes such as the above-referenced urease-free creatinine iminohydrolase if it also could stabilize the enzyme during the process and improve the yield of the desired enzyme.